Neurotransmitter Sensors and Methods of Using the Same

ABSTRACT

Neurotransmitter biosensors are disclosed, including YbeJ-based glutamate binding biosensors, comprising a neurotransmitter binding domain conjugated to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon binding neurotransmitter. Such biosensors are useful for the detection of neurotransmitter concentrations in vivo and in culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is divisional of U.S. patent application Ser. No. 11/665,343, filed Apr. 13, 2007, which is a U.S. National Phase Application of International Application No. PCT/US2005/036956, filed Oct. 14, 2005, which claims the benefit of U.S. Provisional Patent Application 60/618,179, filed Oct. 14, 2004, and U.S. Provisional Patent Application 60/643,576, filed Jan. 14, 2005, which are incorporated herein by reference in their entireties.

This application is also related to provisional application Ser. No. 60/658,141, provisional application Ser. No. 60/658,142, provisional application Ser. No. 60/657,702, PCT application No. PCT/US2005/036955, PCT application No. PCT/US2005/036953, and PCT application no. PCT/US2005/036951, which are herein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded through two grants, including an NIH subcontract from Duke University (Subcontract No. SPSID 126632) and a Human Frontier Science Program grant (Contract No. RGP0041/2004C). This invention was also funded by DOE Grant No. DE-FG02-04ER15542 and by NIH Grant No. 1 R33DK070272. Accordingly the U.S. Government has certain rights to this invention.

FIELD OF INVENTION

The invention relates generally to the field of neurotransmitter signaling and, more specifically, to biosensors and methods for measuring and detecting changes in neurotransmitter levels using fluorescence resonance energy transfer (FRET).

BACKGROUND OF INVENTION

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Glutamate is an amino acid and one type of neurotransmitter found in the brain. Glutamatergic neurons are the predominant excitatory pathways in the mammalian brain, representing one-third of all rapid excitatory synapses in the central nerve system (Cotman, C. W., and Monaghan, D. T. (1986) Anatomical organization of excitatory amino acid receptors and their properties. Adv. Exp. Med. Biol. 203, 237-252). Signaling by glutamate is mediated by a large and diverse number of receptors, including ionotropic receptors that allow passage of extracellular calcium through coupled ion channels upon activation, and metabotropic receptors that activate intermediary molecules such as G proteins to produce molecules such as IP₃ that increase cytosolic calcium concentrations. Interaction between neurons may be either excitatory or inhibitory. The major excitatory amino acid neurotransmitters are glutamate and aspartate, while GABA (γ-aminobutyric acid), glycine (aminoacetic acid), and taurine are inhibitory (Mark et al. (2001) American Journal of Neuroradiology 22:1813-1824).

Clearance of extracellular glutamate by glutamate transporters is an indispensable step to prevent the accumulation of glutamate, which would otherwise result in overstimulation of glutamate receptors and glutamate excitotoxicity. Excitotoxic damage causes, or is involved in, a number of neurologic diseases, including stroke, trauma, epilepsy, and neurodegenerative conditions, such as Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis (Doble, A., Louvel, E., and Hugon, J. (1999) The role of excitotoxicity in neurodegenerative disease: implications for therapy, Pharmacol. Ther. 81(3):163-221; Waggie K S, Kahle P J, Tolwani R J. (1999) Neurons and mechanisms of neuronal death in neurodegenerative diseases: a brief review. Lab. Anim. Sci. 49:358-362). Glutamate receptor overstimulation increases intracellular calcium by directly opening ion channels, allowing the influx of calcium and causing membrane depolarization. Depolarization in turn activates voltage-dependent calcium channels, which further increases the intracellular calcium levels. The glutamate-induced elevated calcium levels causes overactivation of a number of enzymes, including protein kinase C, calcium/calmodulin-dependent protein kinase II, phospholipases, proteases, phosphatases, nitric oxide synthase, endonucleases, and ornithine decarboxylase, some of which produce toxic free oxygen radicals, or produce positive feedback loops leading to neuronal death (Mark et al., 2001).

The key factor that triggers the excitotoxic cascade is the excessive accumulation of glutamate in the synaptic space. Normal extracellular glutamate concentration is about 0.6 μmol/L, with substantial neuronal excitotoxic injury occurring at glutamate concentrations of 2 to 5 μmol/L. Traumatic injury to neurons can produce disastrous results with the release of about 10 μmol/L to the extracellular space. Given the ensuing cascade, injury to a single neuron puts all of the neighboring neurons at risk (Mark et al., 2001).

Despite a number of studies showing the involvement of higher glutamate concentration in neurologic diseases, measuring glutamate concentration in living cells remains challenging. One of the most important tools required to assign functions of neurons in vivo would be to visualize glutamate fluxes directly. The extracellular concentration of glutamate has been measured by in vivo microdialysis techniques (Faden, A. I., Demediuk, P., Panter, S. S., and Vink, R. (1989) The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244, 798-800; Fallgren, A. B., and Paulsen, R. E. (1996) A microdialysis study in rat brain of dihydrokainate, a glutamate uptake inhibitor. Neurochem Res 21, 19-25). However, microdialysis is limited in spatial and temporal resolution, unable to detect the localized and rapid concentration change around a single synapse. In addition, the in vivo microdialysis technique is destructive. It also does not permit direct monitoring of glutamate levels inside living neurons or astrocytes.

In vivo measurement of ions and metabolites by using Fluorescence Resonance Energy Transfer (FRET) has been successfully used to measure calcium concentration changes, by fusing CFP, YFP, and a reporter domain consisting of calmodulin and the M13 peptide (Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002a) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906-918; Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002b) Creating new fluorescent probes for cell biology. Nature Reviews Molecular Cell Biology 3, 906-918). Binding of calcium to calmodulin causes global structural rearrangement of the chimera resulting in a change in FRET intensity as mediated by the donor and acceptor fluorescent moieties. Recently a number of bacterial periplasmic binding proteins, which undergo a venus flytrap-like closure of two lobes upon substrate binding, have been successfully used as the scaffold of metabolite nanosensors (Fehr, M., Frommer, W. B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851; Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127-19133; Lager, I., Fehr, M., Frommer, W. B., and Lalonde, S. (2003) Development of a fluorescent nanosensor for ribose. FEBS Lett 553, 85-89).

In order to develop a nanosensor protein for glutamate, we searched for a protein which changes conformation upon binding glutamate. The family of ionotropic (iGluRs) and metabotropic glutamate receptor (mGluRs) have an extracellular ligand binding domain which has sequence similarity to bacterial periplasmic binding proteins (O'Hara, P. J., Sheppard, P. O., Thogersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron 11, 41-52), as well as the ligand binding domain of γ-aminobytyric acid (GABA)_(B) receptor (Kaupmann, K., Huggel, K., Heid, J., Flor, P. J., Bischoff, S., Mickel, S. J., McMaster, G., Angst, C., Bittiger, H., Froestl, W., and Bettler, B. (1997) Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239-246). The crystal structures of mGluR1 ligand binding domain in three different forms, in a complex with glutamate and in two unliganded forms, has been determined (Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000). Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971-977), and the results suggested that glutamate binding stabilizes the closed conformation. Galvez et al. suggested that the ligand binding domain of GABA_(B) receptor also undergoes the closure of two lobes (Galvez, T., Parmentier, M. L., Joly, C., Malitschek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., and Pin, J. P. (1999). Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding. J Biol Chem 274, 13362-13369). We therefore attempted to construct FRET biosensors using the mGluR and GABA_(B) receptors and assayed for changes in FRET efficiency upon addition of substrates. However, no change in FRET efficiency was observed. Similarly, also the LIV leucine/isoleucine/valine amino acid binding protein from bacteria could not be engineered into a functional FRET sensor.

De Lorimier et al. have shown that the YbeJ protein from E. coli, which shares sequence homology to glutamine- and histidine-binding proteins, and which is located in an operon involved in glutamate metabolism, binds to glutamate and aspartate (de Lorimier, R. M., Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M., Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., and Helling a, H. W. (2002) Construction of a fluorescent biosensor family. Protein Sci 11, 2655-2675). The similarity of YbeJ to glutamine and histidine binding proteins from bacteria lead us to generate homology models based on the solved crystal structures of these two proteins. The 3D structure of the glutamine and histidine binding proteins indicates that N- and C-termini of these proteins are located on the same lobe, therefore the closure of two lobes upon substrate binding is unlikely to change the distance between N- and C-terminus. Thus none of these proteins should permit the construction of a FRET sensor on the same principle. Indeed, the sensors proposed by Helling a and Looger in published U.S. patent application 20040118681 propose conjugating a single fluorophore to a cysteine residue that responds to a conformational change upon ligand binding, in contrast to the dual fluorescent moieties used for FRET.

Nevertheless, the present inventors have surprisingly found that the YbeJ protein of E. coli is an efficient FRET scaffold for detecting glutamate binding, despite the finding that both termini are located on the same lobe of the protein. This is in contrast to the general hypothesis that distance changes are converted to FRET changes.

SUMMARY OF INVENTION

The present invention provides neurotransmitter biosensors for detecting and measuring changes in neurotransmitter concentrations. In particular, the invention provides an isolated nucleic acid encoding a glutamate binding fluorescent indicator (FLIP-E) comprising a glutamate binding protein moiety from Escherichia coli YbeJ wherein the glutamate binding protein moiety is genetically fused to a donor fluorescent protein moiety and an acceptor fluorescent protein moiety, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety. Vectors, including expression vectors, and host cells comprising the inventive nucleic acids are also provided, as well as biosensor proteins encoded by the nucleic acids. Such nucleic acids, vectors, host cells and proteins may be used in methods of detecting changes in neurotransmitter levels and particularly extracellular glutamate levels in neuron samples, and in methods of identifying compounds that modulate glutamate excitotoxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FLIP-E nanosensor constructs used for expression in E. coli (A and B) and neuronal cell culture (C and D).

FIG. 2. Spectra of FLIP-E 600n sensor (fluorescent glutamate nanosensor with a K_(d) for glutamate of 600 nM) at three different concentrations of glutamate: 0 mM (black), at the K_(d) (blue), and at saturation (red). Curves share an isosbestic point at 520 nm.

FIG. 3. A hippocampal cell treated with 1 mg/ml trypsin. Images (A-D) were taken at 10 second intervals. Note that signals on the cell surface largely disappear.

FIG. 4. Emission intensity ratio change in a hippocampal cell expressing FLIP-E 600n sensor. The images are pseudo-colored to indicate the emission intensity ratio change. Open bars above the graph (A) indicate the time point of treatment (stimulation/perfusion with glutamate). Ratio images at the time points indicated by arrows are shown in panel (B), a to i. The change in emission intensity ratio was both observed upon electrical stimulation and upon perfusion with glutamate. The ratio change was not observed when perfusing with low levels of substrate (10 nM glutamate).

FIG. 5. Emission intensity ratio change in a hippocampal cell expressing FLIP-E 10p sensor (fluorescent glutamate nanosensor with a K_(d) for glutamate of 10 μM). Open bars above the graph (A) indicate the time point of treatment (stimulation/perfusion with glutamate). Ratio images at the time points indicated by arrow are shown in panel (B), a to g. Electrical stimulation did not cause a large change in the emission intensity ratio, whereas perfusion with 100 μM glutamate induces a reversible ratio change (panel (B), c and e).

DETAILED DESCRIPTION OF INVENTION

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Other objects, advantages and features of the present invention become apparent to one skilled in the art upon reviewing the specification and the drawings provided herein. Thus, further objects and advantages of the present invention will be clear from the description that follows.

Biosensors

The present invention provides neurotransmitter biosensors for detecting and measuring changes in neurotransmitter concentrations using Fluorescence Resonance Energy Transfer (FRET). The three major categories of substances that act as neurotransmitters are (1) amino acids (primarily glutamic acid or glutamate, GABA, aspartic acid & glycine), (2) peptides (vasopressin, somatostatin, neurotensin, etc.) and (3) monoamines (norepinephrine, dopamine & serotonin) plus acetylcholine. In particular, the invention provides glutamate binding fluorescent indicators, particularly indicators comprising a glutamate binding protein moiety from the Escherichia coli glutamate/aspartate receptor, YbeJ. Additional neurotransmitter biosensors for the neurotransmitters listed above may also be prepared using the constructs and methods provided herein.

Thus, the invention provides isolated nucleic acids encoding neurotransmitter binding fluorescent indicators. One embodiment, among others, is an isolated nucleic acid which encodes a glutamate binding fluorescent indicator, the indicator comprising: a glutamate binding protein moiety, a donor fluorescent protein moiety genetically fused to the glutamate binding protein moiety, and an acceptor fluorescent protein moiety genetically fused to the glutamate binding protein moiety, wherein FRET between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety. A preferred glutamate binding protein moiety is a glutamate binding protein moiety from Escherichia coli YbeJ.

YbeJ is also known in the art as YzzK and GltI, and its DNA sequence (SEQ ID No. 1) and protein sequence (YbeJ, protein accession no NP_(—)415188, SEQ ID No. 2) are known. Any portion of the YbeJ DNA sequence which encodes a glutamate binding region may be used in the nucleic acids of the present invention. For instance, one region that is suitable for use in the nucleic acids of the present invention is provided by SEQ ID No. 3, which encodes a truncated glutamate-aspartate binding protein sequence (SEQ ID No. 4), encoding mature protein without signal peptide. Naturally occurring homologues from other bacterial species may also be used, for instance the PA5082 gene from Pseudomonas aeruginosa, whose gene product is 70% similar to the YbeJ protein from E. coli. Glutamate binding portions of YbeJ or any of its homologues may be cloned into the vectors described herein and screened for activity according to the disclosed assays.

Naturally occurring species variants of YbeJ may also be used, in addition to artificially engineered variants comprising site-specific mutations, deletions or insertions that maintain measurable glutamate binding function. Variant nucleic acid sequences suitable for use in the nucleic acid constructs of the present invention will preferably have at least 70, 75, 80, 85, 90, 95, or 99% similarity or identity to the gene sequence for YbeJ. Suitable variant nucleic acid sequences may also hybridize to the gene for YbeJ under highly stringent hybridization conditions. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Preferred artificial variants of the present invention may exhibit increased or decreased affinity for glutamate, in order to expand the range of concentration that can be measured by YbeJ-based and other glutamate nanosensors. Preferred artificial variants, among others, include glutamate binding regions comprising the mutations A207G, A207P, A207K A207M, A207S, A207C, A207R, A207V, A207L, A207Q, A207T, A207F, A207Y, A207N, A207W, A207H, A207D, and/or S95W. Additional artificial variants showing decreased or increased binding affinity for glutamate may be constructed by random or site-directed mutagenesis and other known mutagenesis techniques, and cloned into the vectors described herein and screened for activity according to the disclosed assays.

The isolated nucleic acids of the invention may incorporate any suitable donor and acceptor fluorescent protein moieties that are capable in combination of serving as donor and acceptor moieties in FRET. Preferred donor and acceptor moieties are selected from the group consisting of GFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), and enhanced variants thereof, with a particularly preferred embodiment provided by the donor/acceptor pair CFP/YFP-Venus, a variant of YFP with improved pH tolerance and maturation time (Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87-90). An alternative is the MiCy/mKO pair with higher pH stability and a larger spectral separation (Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J. 2004 381:307-12). Criteria to consider when selecting donor and acceptor fluorescent moieties are known in the art, for instance as disclosed in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

Also suitable as either a donor or acceptor is native DsRed from a Discosoma species, an ortholog of DsRed from another genus, or a variant of a native DsRed with optimized properties (e.g. a K83M variant or DsRed2 (available from Clontech)). As used herein, the term “variant” is intended to refer to polypeptides with at least about 70%, more preferably at least 75% identity, including at least 80%, 90%, 95% or greater identity to native fluorescent molecules. Many such variants are known in the art, or can be readily prepared by random or directed mutagenesis of native fluorescent molecules (see, for example, Fradkov et al., FEBS Lett. 479:127-130 (2000)).

When the fluorophores of the biosensor contain stretches of similar or related sequence(s), the present inventors have recently discovered that gene silencing may adversely affect expression of the biosensor in certain cells and particularly whole organisms. In such instances, it is possible to modify the fluorophore coding sequences at one or more degenerate or wobble positions of the codons of each fluorophore, such that the nucleic acid sequences of the fluorophores are modified but not the encoded amino acid sequences. Alternative, one or more conservative substitutions that do not adversely affect the function of the fluorophores may also be incorporated. See PCT application [Attorney Docket No. 056100-5054, “Methods of Reducing Repeat-Induced Silencing of Transgene Expression and Improved Fluorescent Biosensors], which is herein incorporated by reference in its entirety.

The invention further provides vectors containing isolated nucleic acid molecules encoding neurotransmitter biosensor polypeptides. Exemplary vectors include vectors derived from a virus, such as a bacteriophage, a baculovirus or a retrovirus, and vectors derived from bacteria or a combination of bacterial sequences and sequences from other organisms, such as a cosmid or a plasmid. Such vectors include expression vectors containing expression control sequences operatively linked to the nucleic acid sequence coding for the neurotransmitter biosensor. Vectors may be adapted for function in a prokaryotic cell, such as E. coli or other bacteria, or a eukaryotic cell, including yeast and animal cells. For instance, the vectors of the invention will generally contain elements such as an origin of replication compatible with the intended host cells, one or more selectable markers compatible with the intended host cells and one or more multiple cloning sites. The choice of particular elements to include in a vector will depend on factors such as the intended host cells, the insert size, whether regulated expression of the inserted sequence is desired, i.e., for instance through the use of an inducible or regulatable promoter, the desired copy number of the vector, the desired selection system, and the like. The factors involved in ensuring compatibility between a host cell and a vector for different applications are well known in the art.

Preferred vectors for use in the present invention will permit cloning of the neurotransmitter binding domain or receptor between nucleic acids encoding donor and acceptor fluorescent molecules, resulting in expression of a chimeric or fusion protein comprising the neurotransmitter binding domain genetically fused to donor and acceptor fluorescent molecules. Exemplary vectors include the bacterial pRSET-FLIP derivatives disclosed in Fehr et al. (2002) (Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851), which is herein incorporated by reference in its entirety. Alternatively, the neurotransmitter binding domain of interest may be first fused to fluorescent donor and acceptor coding sequences and then cloned into an appropriate vector, as described in U.S. Pat. No. 6,596,499, which is herein incorporated by reference in its entirety.

The chimeric nucleic acids of the present invention are preferably constructed such that the donor and acceptor fluorescent moiety coding sequences are fused to separate termini of the neurotransmitter binding domain in a manner such that changes in FRET between donor and acceptor may be detected upon neurotransmitter binding. Alternatively, either or both of the donor fluorophore and/or said acceptor fluorophore moieties may be fused to the ligand binding protein moiety at an internal site of said ligand binding protein moiety. Such fusions are described in provisional application No. 60/658,141, which is herein incorporated by reference. Preferably, the donor and acceptor moieties are not fused in tandem, although the donor and acceptor moieties may be contained on the same protein domain or lobe. A domain is a portion of a protein that performs a particular function and is typically at least about 40 to about 50 amino acids in length. There may be several protein domains contained in a single protein.

Fluorescent domains can optionally be separated from the neurotransmitter binding domain by one or more flexible linker sequences. Such linker moieties are preferably between about 1 and 50 amino acid residues in length, and more preferably between about 1 and 30 amino acid residues. Linker moieties and their applications are well known in the art and described, for example, in U.S. Pat. Nos. 5,998,204 and 5,981,200, and Newton et al., Biochemistry 35:545-553 (1996). Alternatively, shortened versions of the fluorophores or the binding protein may be used.

For instance, the present inventors have also found that removing sequences connecting the core protein structure of the binding domain and the fluorophore, i.e., by removing linker sequences and/or by deleting amino acids from the ends of the analyte binding moiety and/or the fluorophores, closer coupling of fluorophores is achieved leading to higher ratio changes. Preferably, deletions are made by deleting at least one, or at least two, or at least three, or at least four, or at least five, or at least eight, or at least ten, or at least fifteen nucleotides in a nucleic acid construct encoding a FRET biosensor that are located in the regions encoding the linker, or fluorophore, or ligand binding domains. Deletions in different regions may be combined in a single construct to create more than one region demonstrating increased rigidity. Amino acids may also be added or mutated to increase rigidity of the biosensor and improve sensitivity. For instance, by introducing a kink by adding a proline residue or other suitable amino acid. Improved sensitivity may be measured by the ratio change in FRET fluorescence upon ligand binding, and preferably increases by at least a factor of 2 as a result of said deletion(s). See provisional application No. 60/658,141, which is herein incorporated by reference in its entirety.

The invention also includes host cells transfected with a vector or an expression vector of the invention, including prokaryotic cells, such as E. coli or other bacteria, or eukaryotic cells, such as yeast cells or animal cells. In another aspect, the invention features a transgenic non-human animal having a phenotype characterized by expression of the nucleic acid sequence coding for the expression of the neurotransmitter biosensor. The phenotype is conferred by a transgene contained in the somatic and germ cells of the animal, which may be produced by (a) introducing a transgene into a zygote of an animal, the transgene comprising a DNA construct encoding the neurotransmitter biosensor; (b) transplanting the zygote into a pseudopregnant animal; (c) allowing the zygote to develop to term; and (d) identifying at least one transgenic offspring containing the transgene. The step of introducing of the transgene into the embryo can be by introducing an embryonic stem cell containing the transgene into the embryo, or infecting the embryo with a retrovirus containing the transgene. Preferred transgenic animals will express the encoded neurotransmitter biosensor in the brain. Transgenic animals of the invention include transgenic C. elegans and transgenic mice and other animals.

The present invention also encompasses isolated neurotransmitter biosensor molecules having the properties described herein, particularly YbeJ-based glutamate binding fluorescent indicators. Such polypeptides may be recombinantly expressed using the nucleic acid constructs described herein, or produced by chemically coupling some or all of the component domains. The expressed polypeptides can optionally be produced in and/or isolated from a transcription-translation system or from a recombinant cell, by biochemical and/or immunological purification methods known in the art. The polypeptides of the invention can be introduced into a lipid bilayer, such as a cellular membrane extract, or an artificial lipid bilayer (e.g. a liposome vesicle) or nanoparticle.

Methods of Detecting Levels of Neurotransmitters

The nucleic acids and proteins of the present invention are useful for detecting and measuring changes in the levels of neurotransmitters in the brain or nervous system of an animal, particularly changes in the level of extracellular glutamate, which can be a signal of a disorder or disease associated with glutamate excitotoxicity. In one embodiment, the invention comprises a method of detecting changes in the level of extracellular glutamate in a sample of neurons, comprising (a) providing a cell expressing a nucleic acid encoding a glutamate binding biosensor as described herein and a sample of neurons; and (b) detecting a change in FRET between a donor fluorescent protein moiety and an acceptor fluorescent protein moiety, each covalently attached to the glutamate binding domain, wherein a change in FRET between said donor moiety and said acceptor moiety indicates a change in the level of extracellular glutamate in the sample of neurons. Alternatively, the protein may be produced in a heterologous host, e.g. in bacteria, purified and injected into organs directly or into the intercellular spaces. The protein or derivatives thereof may also be coupled to particles including quantum dots and introduced into cells or compartments.

FRET may be measured using a variety of techniques known in the art. For instance, the step of determining FRET may comprise measuring light emitted from the acceptor fluorescent protein moiety. Alternatively, the step of determining FRET may comprise measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety. The step of determining FRET may also comprise measuring the excited state lifetime of the donor moiety or anisotropy changes (Squire A, Verveer P J, Rocks O, Bastiaens P I. J Struct Biol. 2004 July; 147(1):62-9. Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells.). Such methods are known in the art and described generally in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

The amount of glutamate or other neurotransmitter in a sample of neurons can be determined by determining the degree of FRET. First the FLIP-E sensor must be introduced into the sample. Changes in neurotransmitter concentration can be determined by monitoring FRET changes at time intervals. The amount of neurotransmitter in the sample can be quantified for example by using a calibration curve established by titration in vivo.

The neuron sample to be analyzed by the methods of the invention may be contained in vivo, for instance in the measurement of glutamate efflux on the surface of hippocampal neurons, or in vitro, wherein glutamate efflux is measured in neuronal cell culture. Alternatively, a fluid extract from the brain or one or more synaptic spaces may be used as a sample from which extracellular neurotransmitter is detected or measured. Such measurements may be used to detect extracellular glutamate associated with traumatic injury to said neurons, or as a possible indicator of a neurological disorder associated with glutamate excitotoxicity, including stroke, epilepsy, Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis, among others.

Methods for detecting neurotransmitter levels as disclosed herein may be used to screen and identify compounds that may be used to modulate neurotransmitter concentrations and particularly compounds useful for modulating glutamate excitotoxicity. In one embodiment, among others, the invention comprises a method of identifying a compound that modulates glutamate excitotoxicity comprising (a) contacting a cell expressing a glutamate biosensor as disclosed herein and a sample of neurons with one or more test compounds, and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates glutamate excitotoxicity. The term “modulate” means that such compounds may increase or decrease glutamate excitotoxicity. Compounds that increase glutamate levels are targets for therapeutic intervention and treatment of disorders associated with glutamate excitotoxicity, as described above. Compounds that decrease glutamate levels may be developed into therapeutic products for the treatment of disorders associated with glutamate excitotoxicity.

The targeting of the sensor to the outer leaflet of the plasma membrane is only one embodiment of the potential applications. It demonstrates that the nanosensor can be targeted to a specific compartment. Alternatively, other targeting sequences may be used to express the sensors in other compartments such as vesicles, ER, vacuole, etc.

Expression systems comprise not only rat neurons, but also human cell lines, animal cells and organs, fungi and plant cells. The sensors can also be used to monitor levels of glutamate in fungal and plant organisms where glutamate serves as an important nitrogen compound, but potentially also a signaling molecule. Expression in bacteria may be used to monitor glutamate levels at sites of infection or in compartments in which the bacteria reside or are introduced.

Specifically, bacteria or fungi expressing the sensors may serve as biosensors or as tools to identify new pesticides using a similar scheme as outlined for drug screening above.

Additional Utilities

The biosensors of the present invention can also be expressed on the surface of animal cells to determine the function of neurons. For example, in C. elegans, many of the neurons present have not been assigned a specific function. Expression of the biosensors on the surface permits visualization of neuron activity in living worms in response to stimuli, permitting assignment of function and analysis of neuronal networks. Similarly, the introduction of multiphoton probes into the brain of living mice or rats, permits imaging these processes. Finally, expression in specific neurons or glia will allow the study of phenomena such as stroke or Alzheimers Disease and the effect of such disorders on glutamate levels inside neuronal cells or on their surface. Moreover, the effect of medication on localized brain areas or neuronal networks can be studied in vivo.

Finally, it is possible to use the sensors as tools to modify glutamate fluxes by introducing them as artificial glutamate scavengers, for instance presented on membrane or artificial lipid complexes, and thus to manipulate brain or neuron function.

The following examples are provided to describe and illustrate the present invention. As such, they should not be construed to limit the scope of the invention. Those in the art will well appreciate that many other embodiments also fall within the scope of the invention, as it is described hereinabove and in the claims.

EXAMPLES Example 1 Construction of Nucleic Acids and Vectors

A truncated glutamate-aspartate binding protein sequence (SEQ ID No. 4), encoding mature protein without signal peptide, was amplified by PCR using E. coli genomic DNA as a template. The primers used were 5′-ggtaccggaggcgccgcaggcagcacgctggacaaaatc-3′ (SEQ ID No. 5) and 5′-accggtaccggcgccgttcagtgccttgtcattcggttc-3′ (SEQ ID No. 6). The PCR fragment was cloned into the KpnI site of FLIPmal-25μ (Fehr et al. 2002) in pRSET vector (Invitrogen), exchanging the maltose binding protein sequence with the YBEJ sequence. The resulting plasmid was named pRSET-FLIP-E-600n.

To improve the pH and chloride tolerance and maturation of the sensor protein, the fragment containing the enhanced YFP (EYFP, CLONTECH) sequence in pRSET-FLIP-E-600n was replaced with the coding sequence of Venus, a variant of YFP with improved pH tolerance and maturation time (Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87-90). Affinity mutants carrying substitutions A207G, A207P, A207K, A207M, A207S, A207C, A207R, A207V, A207L, A207Q, A207T, A207F, A207Y, A207N, A207W, A207H, A207D, or S95W were created by site-directed mutagenesis (Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367-382).

pRSET-FLIP-E constructs were transferred to E. coli BL21(DE3)Gold (Stratagene) using electroporation (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning. A laboratory manual. (Cold Spring Harbor N.Y.: Cold Spring Harbor Laboratory Press). FLIP-E proteins expressed in BL21(DE3)Gold strain were extracted and purified as previously described (Fehr et al. 2002). For expression in rat primary neuronal cell culture and PC12 cell culture, FLIP-E 600n and −10μ cassettes were cloned into pDisplay (Invitrogen) as follows: XmaI site and SalI site were introduced on the 5′- and 3′-ends of FLIP-E cassette, respectively, by PCR. The primers used were 5′-gagcccgggatggtgagcaagggcgaggag-3′ (SEQ ID No. 7) and 5′-gaggtcgaccttgtacagctcgtccatgccgag-3′ (SEQ ID No. 8). The PCR fragments were sequenced to confirm that there was no additional PCR error, digested with XmaI/SalI, and cloned into the XmaI/SalI sites of the pDisplay vector. Cell cultures were transfected using a modified calcium phosphate transfection protocol (Xia, Z., Dudek, H., Miranti, C. K., and Greenberg, M. E. (1996). Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425-5436) or Lipofectamine (Invitrogen).

Example 2 In Vitro Characterization of FLIP-E Nanosensors

A DNA fragment encoding the mature YBEJ protein was fused to ECFP and the Venus sequence at the N- and C-termini, respectively (FIG. 1). Emission spectra and substrate titration curves were obtained by using monochromator microplate reader Safire (Tecan, Austria). Excitation filter was 433±12 nm, emission filters for CFP and YFP emission were 485±12, 528 nm±12 nm, respectively. All analyses were done in 20 mM sodium phosphate buffer, pH 7.0.

Addition of glutamate resulted in an increase in CFP emission and a decrease in YFP emission, suggesting that binding of glutamate to YBEJ results in a conformational change of the chimeric protein potentially due to a relative change in the orientation of the dipoles of the fluorophores (FIG. 2). Since CFP and YFP moieties are assumed to be attached to the same lobe, we speculate that glutamate binding causes the change in dipole-dipole angle of two fluorophores. Interestingly, the ratio and ratio change were in a similar range as compared to other sensors generated so far (Fehr et al., 2002; Fehr et al., 2003; Lager et al., 2003), suggesting that distance changes may not be the primary factor in underlying the mechanisms for FRET changes. Spectra at three different glutamate concentrations (zero, Kd, saturation) reveals an isosbestic point at 520 nm (FIG. 2). The binding constant (Kd) for glutamate was determined to be 600 nM, consistent with data obtained by other methods (de Lorimier et al., 2002). Binding constants for aspartate, glutamine, asparagine were determined to be 1 μM, 100 μM, 300 μM, respectively (see Table 1, below).

In order to expand the range of concentration that can be measured by YBEJ-based glutamate nanosensors, the YBEJ moiety was mutagenized to create nanosensors with lower affinity for glutamate. It has previously been shown that conjugating various fluorophores to sites located at the perimeter of the interdomain cleft that forms the ligand binding site (named “peristeric”) changes the ligand-binding affinity in periplasmic binding proteins (de Lorimier et al., 2002). Among the residues tested, mutation of alanine 207 to lysine, methionine, serine, cysteine, arginine, valine, leucine, glutamine, threonine, phenylalanine, tyrosine, aspargine, tryptophan, histidine, aspartate lowered the binding affinity significantly (Table 1). In addition, the mutation of serine 118 to tryptophan, which is suggested to interact with the nitrogen of glutamate, was found to decrease the affinity of the protein. Thus, mutations introduced into the FLIPE nanosensor can yield affinity mutants suitable to cover a wide range of physiological glutamate concentrations.

TABLE 1 Kd(M) YbeJ Kd(M) Kd(M) Kd(M) Aspara- Vector moiety Glutamate Aspartate Glutamine gine FLIPE-600n-1 WT 6 × 10⁻⁷ 6 × 10⁻⁶ 1 × 10⁻⁴ 3 × 10⁻⁴ FLIPE-600n-2 A207G 6 × 10⁻⁷ 4 × 10⁻⁶ 2 × 10⁻⁴ n.d. FLIPE-600n-3 A207P 6 × 10⁻⁷ 4 × 10⁻⁶ 2 × 10⁻⁴ n.d. FLIPE-3μ A207K 3 × 10⁻⁶ 2 × 10⁻⁵ 7 × 10⁻⁴ n.d. FLIPE-5μ A207M 5 × 10⁻⁶ 3 × 10⁻⁵ 1 × 10⁻³ n.d. FLIPE-5μ-2 A207S 5 × 10⁻⁶ 3 × 10⁻⁵ 1 × 10⁻³ n.d. FLIPE-6μ A207C 6 × 10⁻⁶ 5 × 10⁻⁵ n.d. n.d. FLIPE-10μ-1 A207R 1 × 10⁻⁵ 6 × 10⁻⁵ 1 × 10⁻³ n.d. FLIPE-10μ-2 A207V 1 × 10⁻⁵ 8 × 10⁻⁵ 6 × 10⁻³ n.d. FLIPE-30μ A207L 3 × 10⁻⁵ n.d. n.d. n.d. FLIPE-40μ-1 A207Q 4 × 10⁻⁵ 2 × 10⁻⁴ 7 × 10⁻³ n.d. FLIPE-40μ-1 A207T 4 × 10⁻⁵ 1 × 10⁻⁴ 7 × 10⁻³ n.d. FLIPE-100μ-1 S95W 1 × 10⁻⁴ n.d. n.d. n.d. FLIPE-100μ-2 A207F 1 × 10⁻⁴ 6 × 10⁻⁴ n.d. n.d. FLIPE-300μ A207Y 3 × 10⁻⁴ 5 × 10⁻⁴ n.d. n.d. FLIPE-400μ A207N 4 × 10⁻⁴ 1 × 10⁻³ n.d. n.d. FLIPE-1m A207W 1 × 10⁻³ n.d. n.d. n.d. FLIPE-2m-1 A207H 2 × 10⁻³ 2 × 10⁻³ n.d. n.d. FLIPE-2m-2 A207D 2 × 10⁻³ 9 × 10⁻⁴ n.d. n.d.

Example 3 In Vivo Characterization of Flip-E

For the in vivo characterization of FLIP-E nanosensors, FLIPE-600n and FLIPE-10μ were cloned into the mammalian expression vector pDisplay (Invitrogen, USA). The pDisplay vector carries a leader sequence which directs the protein to the secretory pathway, and the transmembrane domain which anchors the protein to the plasma membrane, displaying the protein on the extracellular face. Rat hippocampal cells and PC12 cells were transfected with pDisplay FLIPE-600n and -10μ constructs. FRET was imaged 24-48 hours after transfection on a fluorescent microscope (DM IRE2, Leica) with a cooled CoolSnap HQ digital camera (Photometrics). Dual emission intensity ratios were simultaneously recorded following excitation at 436 nm and splitting CFP and Venus emission by DualView with the OI-5-EM filter set (Optical Insights) and Metafluor 6.1r1 software (Universal Imaging).

The expression of FLIP-E was observed on the plasma membrane of rat hippocampal cell culture, and to some extent also in intracellular compartments, probably in compartments involved in plasma membrane targeting of plasma membrane proteins. When treated with Tyrode's buffer containing 1 mg/mL of trypsin, the majority of fluorescence on the cell surface was eliminated, demonstrating that the FLIPE protein was indeed displayed on the extracellular face of the plasma membrane as expected from the properties of the pDisplay construct (FIG. 3). The nanosensors should thus measure extracellular glutamate levels close to the cell's surface.

To quantify the intensity of CFP and Venus emission, the fluorescence intensity in the two channels in the periphery of the cell was integrated on a pixel-by-pixel basis, and the CFP/Venus ratio was calculated. When the hippocampal cells displaying FLIPE-600n on the surface were electrically stimulated by passing current pulse, a decrease in CFP/Venus emission ratio was observed (FIG. 4 a-c), suggesting that the glutamate is released from hippocampal cells by membrane depolarization. To confirm that the ratio change is due to changes in the extracellular concentration of glutamate, the cell was perfused with increasing concentrations of glutamate. The emission intensity ratio changed in a concentration dependent manner, (FIG. 4 d-h), indicating that the FLIPE-600n displayed on the cell surface recognizes the extracellular glutamate. The working range of the FLIP-E 600n sensor was between 100 nM to 1 μM, which is consistent with the in vitro working range of FLIPE-600n nanosensor. The CFP/Venus ratio increased when the external medium was washed away by perfusion, suggesting that the change in FRET intensity in vivo is reversible.

In contrast to the cells expressing FLIPE/600n sensor, the CFP/Venus emission intensity change was not observed in cells expressing FLIPE-10μ upon electro-stimulation (FIG. 5). However, a ratio change was observed when the cells were perfused with higher concentrations of glutamate, (FIG. 5 c and e), suggesting that the glutamate concentration change induced by depolarization of the cell was below the working range of FLIP-E 10μ sensor.

The novel nanosensors are thus able to measure glutamate on the surface of neuronal cells and to follow the glutamate secretion of presynaptic neurons directly.

All publications, patents and patent applications discussed herein are incorporated herein by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. 

1-30. (canceled)
 31. A glutamate binding fluorescent indicator fusion protein comprising: a glutamate binding protein moiety from Escherichia coli YbeJ wherein the glutamate binding protein moiety is truncated; a donor fluorescent protein moiety covalently coupled to the glutamate binding protein moiety; and an acceptor fluorescent protein moiety covalently coupled to the glutamate binding protein moiety; wherein the donor and the acceptor moieties are on the same lobe of the folded glutamate binding fluorescent indicator, and wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety.
 32. The glutamate binding fluorescent indicator fusion protein of claim 31, wherein said glutamate binding protein moiety consists of a protein sequence as set forth in SEQ ID NO:
 4. 33. The glutamate binding fluorescent indicator fusion protein of claim 31, wherein said donor fluorescent protein moiety is selected from the group consisting of a GFP, a CFP, a BFP, a YFP and a dsRED.
 34. The glutamate binding fluorescent indicator fusion protein of claim 31, wherein said acceptor fluorescent protein moiety is selected from the group consisting of a GFP, a CFP, a BFP, a YFP and a dsRED.
 35. The glutamate binding fluorescent indicator fusion protein of claim 31, wherein said donor fluorescent protein moiety is a CFP and said acceptor fluorescent protein moiety is YFP Venus.
 36. The glutamate binding fluorescent indicator fusion protein of claim 31, wherein the glutamate binding fluorescent indicator further comprises at least one linker moiety.
 37. An isolated cell containing the glutamate binding fluorescent indicator fusion protein of claim
 31. 38. The cell of claim 37, wherein the cell is a prokaryote.
 39. The cell of claim 37, wherein the cell is E. coli.
 40. The cell of claim 37, wherein the cell is a eukaryotic cell.
 41. The cell of claim 37, wherein the cell is a yeast cell.
 42. The cell of claim 37, wherein the cell is an animal cell.
 43. The glutamate binding fluorescent indicator fusion protein of claim 31, further comprising one or more amino acid substitutions that lower the affinity of the glutamate binding protein moiety to glutamate.
 44. The glutamate binding fluorescent indicator fusion protein of claim 43, wherein said one or more nucleic acid substitutions result in an altered amino acid in SEQ ID NO: 4, and wherein the altered amino acid is selected from the group consisting of A175K, A175M, A175S, A175R, A175V, A175L, A175Q, A175T, A175F, A175Y, A175N, A175W, A175H, A175D, and S63W.
 45. A method of detecting changes in the level of extracellular glutamate in a sample of neurons, comprising: (a) providing a cell with the fusion protein of claim 31 and a sample of neurons; and (b) detecting a change in FRET between said donor fluorescent protein moiety and said acceptor fluorescent protein moiety, wherein a change in FRET between said donor moiety and said acceptor moiety indicates a change in the level of extracellular glutamate in a sample of neurons.
 46. The method of claim 45, wherein the step of determining FRET comprises measuring light emitted from the acceptor fluorescent protein moiety.
 47. The method of claim 45, wherein determining FRET comprises measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety.
 48. The method of claim 45, wherein the step of determining FRET comprises measuring the excited state lifetime of the donor moiety.
 49. The method of claim 45, wherein said sample of neurons is contained in vivo.
 50. The method of claim 45, wherein said sample of neurons is contained in vitro.
 51. The method of claim 49, wherein said change in the level of extracellular glutamate is caused by traumatic injury to said neurons.
 52. The method of claim 45, wherein said change in the level of extracellular glutamate is associated with a disorder selected from the group consisting of stroke, epilepsy, Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis.
 53. A method of identifying a compound that modulates glutamate excitotoxicity, comprising: (a) contacting a cell containing the fusion protein of claim 31 and a sample of neurons with one or more test compounds; and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates glutamate excitotoxicity. 